In Italy, watermelon cultivation spans 9510 hectares, with production levels largely influenced by seasonal market demand. As a result, surplus watermelon left unsold by September often remain in the fields, where they decompose naturally and go to waste. A chemical analysis of the watermelon liquid fraction waste (WW) indicates a high carbohydrate concentration, highlighting the potential for biotechnological valorization of this waste stream, converting it into lipids or exopolysaccharides (EPSs). This study investigates the feasibility of utilizing WW as an alternative growth substrate for microalgae, aligning with circular economy principles and advancing sustainable agricultural practices. By repurposing agricultural byproducts, this research supports biorefinery objectives, aiming to convert biomass into high-value secondary products, including biofuels, pigments, and nutraceuticals. Scenedesmus and Chlorella strains demonstrated promising growth and adaptability in WW, achieving biomass yields of 0.95 ± 0.07 g L−1 and 0.37 ± 0.02 g L−1, respectively, with a significant EPS production observed as medium gelation. Although lipid accumulation was limited in this case by the WW substrate, the lipid profiles of both strains were distinctively altered, notably lacking linolenic acid.